The present invention relates to photoreleasable compounds, to processes for making and purifying these compounds and to their uses.
Photocleavable (caged) reagents that release biologically active compounds rapidly upon flash irradiation with near-UV light are potentially valuable tools for study of biological processes (1). However, reagents that can photorelease effectors such as neuroactive amino acids rapidly and efficiently in aqueous solution have proved elusive. Thus, while there are reports in the prior art of approaches to the problem based on a wide range of different photolabile protecting groups (2), overall these have met with, limited success. Among the better current reagents are the p-hydroxyphenacyl esters described by Givens et al (3), but even these are susceptible to hydrolysis and have poor efficiency of photorelease because of their low extinction coefficient in the 300-350 nm region. A very recent report (4) describes photorelease of L-glutamate from brominated 7-hydroxycoumarin-4-ylmethyl esters and carbamates but these respectively are susceptible to hydrolysis or are rate-limited by decarboxylation of the photochemically-generated N-carboxyglutamate (k=xcx9c150 sxe2x88x921 at pH 7,21xc2x0 C.) (5).
Pass et al have also reported the use of 5-bromo-7-nitroindolinyl (Bni) as a protecting group during photochemical peptide synthesis to protect carboxylic groups and activate them on irradiation to couple to other nucleophiles (6b).
Broadly, the present invention relates to photoreleasable compounds comprising a caging moiety linked to an effector moiety, wherein the compounds are capable of releasing the effector moiety on irradiation, typically by flash irradiation with UV light. The photoreleasable compounds can therefore be used to deliver biologically active effector moieties such as neuroactive amino acids or metal chelators to sites where their activity is required. In preferred embodiments of the invention, the caging moiety is based on 7-nitroindoline and substituted derivatives thereof.
Accordingly, in one aspect, the present invention provides a compound represented by the structural formula: 
wherein
R1 is hydrogen;
C1-10 alkyl or substituted alkyl;
O(CH2)nxe2x80x94Y;
N(COZ)(CH2)mY; or
N[(CH2)mX][(CH2)nY];
R2 and R3 are independently selected from:
hydrogen;
C1-10 alkyl or substituted alkyl; or
R2 and R3 together are cycloalkyl;
R4 is hydrogen;
C1-10 alkyl or substituted alkyl;
phenyl or substituted phenyl;
(CH2)nY; or
(CH2)mO(CH2)nY;
wherein:
m and n are independently between 1 and 10;
X and Y are independently selected from hydrogen, CO2H or salts thereof or OPO32xe2x88x92;
Z is hydrogen or C1-10 alkyl or substituted alkyl; and,
X is an effector moiety or a group capable of being coupled or converted to an effector moeity.
In one embodiment, the present invention provides compounds represented by the structural formula: 
wherein
R2 and R3 are independently selected from hydrogen C1-10 alkyl or substituted alkyl, or R2 and R3 together are cycloalkyl;
R4xe2x80x2 is a blocking group; and,
X is an effector moiety.
The R4xe2x80x2 group blocks the 5-position to ensure that the nitration reaction occurs at the 7-position of the indoline ring. Preferably, R4xe2x80x2 is selected from:
hydrogen;
C1-C10 alkyl or substituted alkyl;
phenyl or substituted phenyl;
(CH2)nCO2Y; and,
(CH2)nxe2x80x94Oxe2x80x94(CH2)mY;
wherein:
m and n are independently between 0 and 10; and,
Y is hydrogen, or C1-C10 alkyl or substituted alkyl.
Exemplary compounds of the invention include:
Methyl 1-glutaryl-7-nitroindoline-5-acetate 8;
Methyl 1-[(5-dihydroxyphosphoryloxy)pentanoyl)]-7-nitroindoline-5-acetate 9;
Methyl 1-[S-(4-amino-4-carboxybutanoyl)]-7-nitroindoline-5-acetate 10;
Methyl 1-(4-aminobutanoyl)-7-nitroindoline-5-acetate 21;
Methyl 1-acetyl-7-nitroindoline-5-acetate 16;
Mono[1-(5-methoxycarbonylmethyl-7-nitroindolyl)] amide of 1,2-bis(O-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid;
1-Acetyl-4-methoxy-7-nitroindoline 25;
1-Acetyl-4-methoxy-5-methyl-7-nitroindoline 25;
1-[S-(4-Amino-4-carboxybutanoyl)]-4-methoxy-7-nitroindoline;
1-(4-Aminobutanoyl)-4-methoxy-7-nitroindoline;
1-(5-Dihydroxyphosphoryloxy)pentanoyl))-4-methoxy-7-nitroindoline;
Mono[1-(4-methoxy-7-nitroindolyl)] amide of 1,2-bis(O-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid;
1-[S-(4-Amino-4-carboxybutanoyl)]-4-methoxy-5-methyl-7-nitroindoline;
1-(4-Aminobutanoyl)-4-methoxy-5-methyl-7-nitroindoline;
1-[(5-Dihydroxyphosphoryloxy)pentanoyl)]-4-methoxy-5-methyl-7-nitroindoline; and
Mono[1-(4-methoxy-5-methyl-7-nitroindolyl)] amide of 1,2-bis(O-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid.
In some embodiments of the invention, the caging moiety is based on substituted 7-nitroindoline. Examples showing the synthesis of substituted 7-nitroindolinyl glutamate and substituted 7-nitroindolinyl GABA, and the photorelease of L-glutamate and GABA from these compounds is described in the experimental section below.
Preferably, the photoreleasable compound is not an activator or an inhibitor of a biological process mediated by the effector moiety, i.e. the biological activity of the effector is only released when the compound is exposed to electromagnetic radiation (preferably having a wavelength between 300-350 nm), thereby allowing its controlled delivery. Otherwise, it is preferable that (a) the photoreleasable compound is water soluble and stable to hydrolysis and that (b) the release reaction is preferably fast and proceed with high quantum efficiency.
The photoreleasable compounds of the invention have a variety of uses in studying systems that respond to the effector moiety and in the treatment of conditions which respond to it.
In a further aspect, the present invention provides the above compounds for use in a method of medical treatment.
In a further aspect, the present invention provides the use of the above compounds for the preparation of a medicament for the treatment of a condition which responds to the effector moiety.
In a further aspect, the present invention provides a process for releasing an effector moiety, the process comprising irradiating a photoreleasable compound as described above to cause the release of the effector moiety.
In a further aspect, the present invention provides a process for producing one of the above photoreleasable caged compounds, the process comprising:
(a) reacting an indoline to provide a blocking group at the 5-position;
(b) reacting the indoline of step (a) to couple a effector moiety at the heterocyclic nitrogen, the effector group having a protecting group; and,
(c) nitrating the indoline of step (b) at the 7-position.
The sequence of steps specified above has the advantage of avoiding a difficult acylation reaction of 7-nitroindolines. This is advantageous as the conditions required for such a reaction can be detrimental for effector moieties with sensitive side chains such as glutamate.
Conveniently, the protecting group can be selected so that the nitration reaction concurrently removes the protecting group from the effector moiety. This has the advantage that protecting groups present on the effector moiety are concurrently removed in the nitration reaction,
In a further aspect, the present invention provides a process for purifying a photoreleasable compound as described above, optionally after producing it as indicated above, the process comprising:
(a) eluting the caged compound from a HPLC column using aqueous methanol containing buffer salts;
(b) desalting fractions containing the caged compound obtained from step (a) on Amberlite XAD-2 resin;
(c) eluting the resin with methanol to recover the caged compound.
The use of this purification procedure to recover the substituted 7-nitroindolinyl GABA is shown in the examples below and is particularly applicable to the purification of photoreleasable compounds having hydrophobic effector groups.
Embodiments of the present invention will now be described by way of example and not by limitation with reference to the accompanying drawings.